Protease-mediated drug delivery: Investigation of photosensitizer peptide conjugates for increased PDT efficacy

Protease-mediated Photodynamic Therapy (PDT) drug delivery uses a tricomponent drug that includes a photosensitizer (PS), a solubilizer, and a peptide linkage that is cleaved by specific enzymes that are over-expressed by tumours. Two PS-peptide conjugates were examined as model compounds for investigating the application of this novel drug delivery method to PDT. The first model compound used hematoporphyrin IX (HpIX) as the PS, tetraarginine as the solubilizer and a small peptide chain sensitive to chymotrypsin. This first model compound was called HpIX-conjugate. The second model compound used pyropheophorbide-a (Ppa) as the PS and poly-L-lysine, which acted as the solubilizer and the enzyme-specific linker sensitive to trypsin (Ppa-conjugate) The photophysical and pharmacological properties of these drug conjugates were characterized and compared to their respective parent PS.;In vitro studies revealed that both conjugated PSs exhibited a significant dark toxicity when compared to their respective parent compounds HptX exhibited no dark toxicity at the concentrations used (0.1-10 muM), whereas HpIX-conjugate had an IC50 = 0.12 muM. Similarly, Ppa did not present any dark toxicity at the concentrations used (0.110 muM); the Ppa-conjugate had an IC50 = 0.52 muM. Further in vitro studies investigated the sub-cellular localization of these compounds and it was found that HpIX localized in the mitochondria, the lysosome and in the cytoplasm, whereas the HpIX-conjugate was unable to enter the cell and was only observed in the cell membrane. However, the Ppa and the Ppa-conjugate are both capable of entering the cells and localized in the endoplasmic reticulum/golgi apparatus or cytoplasm.;The results of these studies on PDT peptide drug conjugates have demonstrated that the addition of small peptides (HpIX-conjugate) or macromolecules (Ppa-conjugate) does not alter the intrinsic PS fluorescence and absorbance of the PS, but has a deleterious effect on dark toxicity. In addition, the investigation revealed that conjugating small peptides to PS (HpIX-conjugate) does not affect the photosensitization effect of the PS, as opposed to conjugation with the macromolecule (Ppa-conjugate), which quenched the effect of the PS. It was further determined that the size of the peptide conjugate dictated the internalization of the PS, as small peptides precluded the entry of the PDT conjugates while macromolecular PSs were internalized.;Photophysical characterization of these compounds demonstrated that conjugation of a small peptide or macromolecule to PSs does not change their intrinsic fluorescence and absorbance properties and that the singlet oxygen quantum yields of the HpIX and the HpIX-conjugate were overlapping (&phis;HpIX = 0.40: &phis;HpIX-conjugate = 0.28). On the other hand, the quantum yield of Ppa was substantially reduced upon conjugation to the polylysine chain (&phis;Ppa = 0.49; &phis;PPa-conjugate = 0.16).